Studies by Shibata et al (1-4) have shown that oral delivery of 1-10 μm phagocytosable chitin particles results in an elevation of Th1 cytokines in mouse spleen cell cultures. The effect was specific to the particulates as no elevation was produced by soluble chitin. It could also be reproduced in 1 μm polystyrene microspheres coated with N-Acetyl-D-Glucosamine, which is the main component of chitin. It was also demonstrated that oral administration of chitin down-regulates serum IgE and lung eosinophilia in a murine model of ragweed allergy (1).
Shibata et al have also developed a mouse model of allergic airway inflammation and orally administered chitin preparations to the mice (Shibata 2000). Ragweed-specific IgE levels were significantly reduced after daily oral administration of chitin to ragweed-sensitised mice, before and during immunisation.
When chitin was administered prophylactically to mice who were subsequently administered ragweed, IL-4, IL-5 and IL-10 production was significantly reduced and low but significant levels of IFN-γ were detected.
Chitin also has a prophylactic effect when administered to C57BL6 mice, which are higher responders for cell-mediated immunity/Th1 responses, but lower responders for allergic responses compared with BALB/c mice. When ragweed-sensitised mice were treated simultaneously with ragweed and chitin, the levels of IL-4, IL-5 and IL-10 produced were significantly reduced compared to those stimulated by ragweed alone.
In Applicants' earlier application, WO 03/015744, Applicants described experiments in mice which demonstrated that a suspension of CMP in saline administered intranasally has beneficial immune modulating properties, which can be applied for the treatment of allergic disease and can enhance protection by up-regulation of mechanisms of innate immunity against viral and bacterial infections of the respiratory tract. The beneficial immune regulating properties can also be applied for the treatment of conditions that would benefit from an up-regulation of natural killer (NK) cell activity and/or the secretion of interferon-γ (IFN-γ), such as the treatment of cancer.
In Applicants' earlier application, U.S. 60/815,074, Applicants described the use of CMP as an adjuvant in vaccine compositions. In particular, CMP compositions were found to be capable of synergistically enhancing the protection raised against an antigen from an infectious agent when the CMP compositions were combined with a further adjuvant, such as the cholera toxin B subunit (CTB).
The evidence to date supports the concept that for CMP to be optimally effective the particles should be micronized and fall in the range of 1 to 20 μm. The primary mode of action of CMP depends on phagocytosis by macrophages and other phagocytes and consequently the CMP need to be of a phagocytosable size, which is generally considered to be in the range of 1 to 20 μm.
Chitin is manufactured from shrimp shell waste, which is a by-product of the shrimp processing industry. The shells are treated with acid and alkali to remove minerals and protein contaminants. The purified chitin flakes are then milled by standard milling methods to produce a powder with a particle size approximately between 100 to 200 μm. This is the starting material for the production of CMP.
In the methods described by Shibata and Strong (3, 5) chitin powder was micronized by sonication in the laboratory. However, this method has the disadvantage of not being amenable for scale-up to industrial manufacturing needs and there are no suitable Good Manufacturing Practice (GMP) protocols established for the micronization of insoluble polysaccharides such as chitin. There is also the further problem that the usual use of sonication is for disruption of agglomerates and cell shearing and sonication does not produce uniform sized particles of CMP.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.